Transcript A novel approach to selectively amplify mRNA in challenging samples “ExpressArt” The University of Vermont Burlington August 22, 2008 Dr.

A novel approach to selectively amplify mRNA in challenging samples
“ExpressArt”
The University of Vermont
Burlington
August 22, 2008
Dr. Guido Krupp
AmpTec
Amplification Technologies
The ExpressArt Technology
FIRST → TRinucleotide priming versus conventional Eberwine technology
for small and divergent sample sizes
• Microarrays and qRT-PCR
• Laser Microdissection, FACS-sorted cells
• “Helpers“ for RNA recovery
SECOND → TRinucleotide priming for reverse transcription: intact and degraded RNAs
with selection for mRNAs and against rRNAs
• FFPE samples with “Helpers“ for RNA recovery
• Exon Arrays: Poor RNA quality with very small samples
THIRD → TRinucleotide priming for reverse transcription: bacterial RNAs
with selection for mRNAs and against rRNAs
• Selective lysis of intracellular bacteria
Why linear amplification?
Amplification of mixed DNA templates with equimolar amounts
• Size matters
• Abundance matters
Exponential, cyclic amplification
per PCR
Mix
1
2
3
4
M
Random Variability
1% per template generation
Thousandfold in 10 cycles: 0.9910 or 0.90
Millionfold in 20 cycles: 0.9920 or 0.82
Linear amplification
by in vitro transcription
Mix Mix
M
1 12
23
34
4M
Random Variability
1% per template generation
Thousandfold in 1-round: 0.99
Millionfold in 2-rounds: 0.992 or 0.9801
mRNA amplification technologies
Problems with very small samples
M
“Eberwine Method”
with one and two amplification rounds
ts
l: uc
o
d
r
nt pro
o
c r
ds
r t i ve i m e
e
n
k
u
r
ar ga p
ro
M ne no
3
2
AmpTec ExpressArt® Technology*
with two and three amplification rounds
ro
un
ds
* Courtesy: Baugh & Hunter, Harvard University
kb
3.0
0.5
0.2
Eberwine: problems with high molecular weight artefacts
Eberwine method
Assessing cRNA Qualities
2 Amplification rounds, 2 ng input RNA
ExpressArt® Technology
2-rounds amplified RNA
18S rRNA position as marker
3-rounds amplified RNA
Mike Mallamaci, AstraZeneca
Eberwine Scheme I:
Synthesis of DNA template for T7 RNA Polymerase
Reverse transcription of mRNA
With Oligo(dT)-T7 primer
T7
Limited RNase H digestion
RNA fragments as primers in
second strand cDNA synthesis
T7
Eberwine Scheme II:
Transcription for first mRNA amplification round
T7
Eberwine Scheme III:
Second round synthesis of DNA template
Problems with divergent sample sizes
High amount of template
Low primer excess
Low amount of template
High primer excess
Random primers
in cDNA synthesis
AAAAA 3´
AAAAA 3´
AAAAA
TTTTT
AAAAA
TTTTT
T7
3´-BIAS: Functional T7 promotor only in 3´-terminal cDNA templates
T7
Eberwine Scheme IV:
Transcription for second mRNA amplification round
T
7
Short antisense RNAs
preferrentially
representing the
3‘-proximal section of
mRNAs
ExpressArt Scheme I:
Synthesis of ds cDNA
T
T
T
T
T
5
’
ds DNA with TRinucleotide-Primer-Mix
Including a 5‘ Box sequence
B
TTTTT-5’
AAAAA-3’
B
TTTTT-5’
AAAAA-3’
“Full-length“
products
ExpressArt Scheme II:
Function of TRinucleotide Primers
21-mer Box + 6-mer Random + Trinucleotide Mix
Box
AGA
TCT
AGA
TCT
TTTTT-5’
Box
Box
AGA AGA
TCT TCT
Box
Box
AGA
TCT
AGA AGA
AGA
TTTTT-5’
AAAAA-3’
ExpressArt: Amplification of a model transcript
Input RNA
800 nt mRNA
Amplified RNA
ExpressArt TRinucleotide primer
ExpressArt: Amplification of a model transcript
Amplified RNA
random primer
Amplified RNA
ExpressArt TRinucleotide primer
ExpressArt Scheme III:
Synthesis of DNA template and transcription for first round
B
TTTTT-5’
AAAAA-3’
First AMPLIFICATION
B
AAAAA-3’
dsDNA with oligo(dT)-T7 primer
B
AAAAA
T TT TT
T7
in vitro transcription
B
U U U U U -5 ’
B
B
U U U U U -5 ’
B
U U U U U -5 ’
U U U U U -5 ’
ExpressArt Scheme IV:
DNA template and transcription for second Round
B
U U U U U -5 ’
1) Reverse transcription of amplified RNA
with Box-Primer
2) RNase to destroy RNA
3) dsDNA with Oligo(dT)-T7-Primer
AAAAA
T TT TT
B
T7
i
n
v
i
t
r
o
t
r
a
n
s
c
r
i
p
t
i
o
n
B
U U U U U -5 ’
B
B
U U U U U -5 ’
B
U U U U U -5 ’
U U U U U -5 ’
Second AMPLIFICATION
ExpressArt Results:
Reproducibility & Comparability
High concordance of microarray data with divergent sample sizes
ExpressArt®
Eberwine*
r=0.994
PCR-coupled amplification*
r=0.979*
r=0.935*
2 rounds:
100ng versus 1.5ng
2 rounds:
50ng versus 10ng
Roche Kit: PCR + IVT
50ng versus 10ng
*Reference: Klur et al. (2004) Genomics 83, 508-517
Evaluation of ExpressArt mRNA amplification kits
starting with
high (4 µg) and very low amounts (2ng) of input total RNA
& comparing one, two and three amplification rounds
Using high quality mouse liver and kidney total RNAs in triplicate experiments
*************************************
Bodo Brunner, Ph.D.
Group sanofi-aventis
Aventis Pharma Deutschland GmbH
Mean foldchange ExpressArt 2 ng 3x IVT
Global foldchange analysis kidney vs. liver (4)
1000
100
r = 0.983
10
1
0.1
0.01
0.001
0.001
0.01
0.1
1
10
100
Mean foldchange ExpressArt 20ng 2x IVT
1000
ExpressArt kit for mRNA Amplification
Pico-version
Ryan Baugh, Kate Hill-Harfe, Gene Brown and Craig Hunter
Dept. of Molecular and Cellular Biology, Harvard University
Expression Profiling Sciences, Wyeth Research
25 June 2003
Genomic Analysis of Embryonic Gene Expression in C. elegans
4-cell-embryo
The nematode Caenorhabditis elegans
Ryan Baugh, Ph.D. Thesis, Harvard, 2003
Yanai, Baugh et al. (2008) Mol. Syst. Biol. 4:163
Descriptive Statistics
Standard_1 Standard_2 Baugh_2ng_1
Average Signal
19,5
18,4
29,2
Median Signal
2
2
4
Max Signal
1777
1538
1615
Present Calls
2964
2926
3373
44.8%
44.2%
50.9%
Baugh_2ng_2
16,9
2
1129
2995
44.7%
Pico_1
20,8
2
2312
3639
Pico_2
20,0
3
1737
3636
55.0%
54.9%
Total number of genes on C.elegans Affymetrix Gene Chip: 6617
Standard
10µg total RNA from Jurkat & H9
cells
hybridization with HG-U133A
2-rounds ExpressArt
100ng total RNA from Jurkat & H9
cells
hybridization with HG-U133A
Presence calls: 9516 (42.8%)
Presence calls: 10149 (45.7%)
Additional genes: 633
Median mRNA length: 4100 nucleotides
Heat map of correlation matrix
1
5x 10-embryos
Baugh
3x 1
1-embryo
embryo
(4-cells)
2x
embryos
2x 10
10-embryos
Yanai, Baugh et al. (2008) Pairing of competitive and topologically distinct
regulatory modules enhances patterned gene expression.
Molecular Systems Biology 4: 163
“We used RNAi and time series, whole-genome microarray analyses to
systematically perturb and characterize components of a Caenorhabditis
elegans lineage-specific transcriptional regulatory network.”
Heat your samples: 2 min @ 70°C
Watch out for your carrier!
11
RNA without heating
10
9
8
7
Fluorescence
6
5
4
3
2
18 S
0
28 S
1
-1
19
24
29
34
39
44
49
54
59
64
69
49
54
59
64
69
Time (seconds)
30
RNA with heating step
25
20
Fluorescence
15
10
18 S
0
19
24
29
34
39
28 S
5
44
Time (seconds)
First “Helper“ for RNA recovery: N-Carrier
Total RNA – microdissected sample with 300 mouse liver cells
--
a 10% aliquot: appr. 0.3 ng
--
ExpressArt N-carrier added: 100 ng (initially)
Second “Helper“ for RNA recovery: NucleoGuard
RNA profiles: blue without
red with NucleoGuard
“The Wong Lab At the UCLA School of Dentistry“
Hu et al., 2008
Exon-level expression profiling: a comprehensive transcriptome analysis for oral fluids.
Clinical Chemistry 54:5.
Second “Helper“ for RNA recovery: NucleoGuard
RNA profiles: blue without
red with NucleoGuard
Dr. Rosemarie Walter
Research Lab at Asterand plc, Detroit, USA
Stoinski-Bangs et al. 2005
Stoinski-Bangs et al. 2005
CellCut®
APPLICATION NOTE
artus GmbH - MMI AG
APPLICATION NOTE
artus GmbH - MMI AG
LASER MICRODISSECTION
ExpressArt®
mRNA AMPLIFICATION
MMI AG
www.mmi-micro.com
artus GmbH
www.artus-biotech.com
1
Amplification Kits for LCM samples
“Helpers“ for LCM samples
Campean et al., 2008. Am J Physiol Renal Baumforth et al., 2008. Am. J. Pathol. 173:195-204.
Birgersdotter et al., 2007. Leuk. Lymph. 48: 2042-2053.
Physiol 294: F1174-84.
Su et al., 2008. J Immunol 181:1264–1271. Bose et al., 2007. J. Pathol. 213:329-336.
Wetzel et al., 2006. Kidney Int. 70: 717-723. Reis et al., 2006. J. Mol. Histol. 37: 79-86.
Okuducu et al., 2005. Int. J. Oncol. 27: 1273-1282.
Users: Novartis, Basel, Switzerland
Asterand, Detroit, USA
Amplification Kits for FACS-sorted cells
“Helpers“ for FACS-sorted cells
”germinal centre (GC) B cells”
Zeng et al., 2005. EMBO J. 23: 4116-4125.
Vockerodt et al., 2008. J. Pathol. 216:83–92.
Differential gene expression by qRT-PCR analysis
Comparison of non-amplified total RNA and ExpressArt amplified RNA (2 rounds)
3
ID3
75
total-RNA
aRNA
Fold ID3 expression
related to case 18428
Fold of Pax5 expression
related to case 18428
PAX5
2
1
total RNA
aRNA
50
25
0
0
18428
18403
18376
18425
18428
18431
18403
300
250
200
150
100
50
18425
18431
STAT6
total RNA
aRNA
Fold STAT6 expression
related to case 18428
Fold AICDA expression
related to case 18428
AICDA
18376
8
6
4
2
0
3
total RNA: no CT(STAT6) measured
aRNA
2
1
0
18428
18403
18376
18425
18431
Dr. Monika Sczepanowski
Centre for Applied Cancer Research
University Clinic Schleswig-Holstein, Kiel, Germany
18428
18403
18376
18425
18431
SECOND → TRinucleotide priming for reverse transcription
Model system for degraded RNA
Controlled cleavage without loss of sequence information
AAAAAA
Fragmentation reaction
Metal-catalyzed cleavage at elevated pH & temperature
AAAAAA
A
Is it possible to recover the complete sequence informations from all fragments?
Function of Box-Random-TRinucleotide-Primer-Mix
Box
AGA
TCT
AGA
TCT
TTTTT-5’
Box
Box
AGA AGA
TCT TCT
Box
Box
AGA
TCT
AGA AGA
AGA
TTTTT-5’
AAAAA-3’
AAAAAA
First cDNA synthesis
A
Preferential priming near the 3’-end of any
nucleic acid sequence
AAAAAA
A
RNA removal
Box
Box2
Second cDNA synthesis
Preferential priming near the 3’-end
Box
Third DNA synthesis
Box-T7-promotor primer
Box2
Box T7
Box2
Box T7
In vitro transcription & amplification
Box2
Box2
Box2
Second
Round
Box2
Box
Box
Box
Box
Amplified antisense RNA
Reverse transcription with Box2 primer
RNA removal
DNA synthesis with Box-T7-promotor primer
Box2
Box T7
RIN = 9.5
Hybridisation Results
Affymetrix GeneChips HG-U133A
Presence Calls
rRNAs [%]
9,794 [:= 100%]
3’-5’-ratios
GAP-DH ß-actin
3.2
1.6
98%
2.8
1.1
2%
98%
2.0
0.9
1.9%
96%
2.5
0.9
1.8%
1.8%
RIN = 3.8
RIN = 3.1
RIN = 2.2
cDNA with Oligo(dT)
82%
>15
>50
0.4%
M:absent
5’:absent
TRinucleotide mRNA Amplification
Intact versus
Severely Degraded RNA
r = 0.993
TRinucleotide mRNA Amplification
Intact versus
Severely Degraded RNA
r = 0.993
Standard mRNA amplification
with Oligo(dT)
r = 0.793
Compact Structure of ribosomal RNAs
ExpressArt® - The future of mRNA amplification
RNA from FFPE Samples
&
High-Quality Microarrays
“Helpers“ for FFPE RNA recovery
DCL (DeCrossLinker) & NG (NucleoGuard)
Useful as template in RT-qPCR:
D Ct = 4 – 10
No additives
Lysis in presence of DCL & NG
Dr. Rosemarie Walter
Research Lab at Asterand plc, Detroit, USA
Whole Transcript ST Arrays
mRNA Amplification without rRNA Depletion
Nano Procedure
(AmpTec)
Starting Material
Minimum of 5 ng total RNA
Intact OR Degraded
Standard Procedure
(Affymetrix)
Micro Procedure
(AmpTec)
Starting Material
Minimum of 1-2 µg total RNA
Starting Material
Minimum of 0.5 µg total RNA
Intact ONLY
Intact OR Degraded
rRNA depletion
Enriched mRNA
300-600 ng
1st double stranded
DNA synthesis
double stranded
template DNA
1st double stranded
DNA synthesis
TRinucleotide Priming is
Selective against rRNA
double stranded
template DNA
1st double stranded
DNA synthesis
TRinucleotide Priming is
Selective against rRNA
1st double stranded
template DNA
1st IVT Amplification with
T7 RNA polymerase
1st cRNA
2nd double stranded
DNA synthesis
2nd double stranded
template DNA
IVT Amplification with
T7 RNA polymerase
> 7 µg cRNA
GeneChip® WT
Double-Stranded cDNA Synthesis Kit
2nd Cycle Double Stranded
cDNA synthesis
> 7.5 µg dsDNA (with dUTP)
GeneChip® WT
Terminal Labeling Kit
Fragmentation &
Labelling
Fragmented / Labelled DNA
Hybridisation to Affymetrix Exon ST Arrays or Gene ST Arrays
EXON ARRAYS
Quality of Input RNA Samples and Overview of Hybridisation Results
Model experiment. Intact RNA samples were chemically degraded, to maintain full sequence complexity in degraded RNA samples
Intact RNA
Degraded RNA
“1000 nt“
Severely
Degraded
RNA
Severely
Degraded
RNA
“500 nt“
“500 nt“
Overview of Hybridisation Results
Method for
generating
cRNAs
Standard
Affymetrix
AmpTec
Starting material
RNA amount /
RNA quality
2.0 µg / intact
cRNA
yields
[µg]
21 ± 5
Sensitivity Mean Signal vs
[% P]
Background
(Ratio)
51 ± 1
280 vs 310
(0.9)
50 ng / intact
2 rounds* 64 ± 2
360 vs 210
TRinucleotide
62 ± 10
(1.7)
AmpTec
50 ng / degraded
2 rounds* 53 ± 2
280 vs 200
TRinucleotide ("1000nt")
58 ± 10
(1.5)
AmpTec
50 ng / severely
2 rounds* 47 ± 3
265 vs 250
TRinucleotide degraded ("500nt") 52 ± 5
(1.1)
Biological
replicates
[Pearson values]
0.98
0.98
0.96
0.95
ROC
pos-neg
cont
0.881
± 0.003
0.901
± 0.004
0.892
± 0.004
0.875
± 0.003
Signal Intensities for Exon Probes over the Complete Length of GAP-DH mRNA
16
14
12
Signal
10
Affy Standard
TR Intact
TR Degraded
TR 75" Degraded
8
6
4
2
0
1
2
3
4
5
Exon
6
7
8
9
Extreme Samples: Low amounts (< 4 ng) of severely degraded saliva RNA
“The Wong Lab At the UCLA School of Dentistry“
Hu et al., 2008
Exon-level expression profiling: a comprehensive transcriptome analysis for oral fluids.
Clinical Chemistry 54:5.
Selective Amplification of Bacterial mRNA
Selective lysis for highly enriched bacterial RNA - Intracellular Listeria and Salmonella
“The Chakraborty lab“
Medical Microbiology, University Giessen, Germany
Original publication: Eriksson et al. (2003) Molecular Microbiology 47: 103–118
“At each time point, infected macrophages were lysed on ice for 30 min in 0.1% SDS, 1%
acidic phenol, 19% ethanol in water. The phenol-ethanol mixture acted to stabilize all bacterial
RNA”
ExpressArt Selective Amplification of Bacterial mRNA
First cDNA synthesis
A
Preferential priming near the 3’-end of any
nucleic acid sequence
A
RNA removal
Box
Box2
Box2
1 ng total RNA
Second cDNA synthesis
Preferential priming near the 3’-end
Box
Third DNA synthesis
Box-T7-promotor primer
Box T7
2 amplification
rounds
> 50 µg amplified RNA
Agilent Bioanalyzer electropherograms of
2-rounds amplified E. coli mRNA
Hybridisation results
Affymetrix E. coli Genome 2.0 GeneChips
38.6% (37°C) or 46.3% (50°C) Presence Calls
Signal-background ratios: 45 - 50.
Scale factors of 10 to 11
Average signals (P)>4,000
Suppression of rRNA amplification:
less than 2% rRNAs in amplified RNAs
Differential gene expression in E. coli: Heat shock
RNAs from E.coli grown at 37°C versus 50°C
Hybridisation with THE MWG E.coli K12 Array
averaged ratios of two dye swap slides
Without amplification
Input 50 µg E.coli RNA
Labelled cDNA with random priming
Hybridisation: 3µg labelled cDNA with
High stringency
ExpressArt mRNA Amplification
Input 100 ng E.coli RNA
Hybridisation: 10µg labelled aRNA
with standard conditions
genes induced during heat shock
genes repressed during heat shock
David Wong
John R. Arrand
Institute for Dentistry
University of California, UCLA
Institute for CancerStudies
University of Birmingham,UK
ALL-EXON-ARRAYS: Degraded RNAs in Saliva
Large and Small Samples
Degraded RNA Samples
Estelle Marrer & Isabelle Keller
Veska Uzunova & Luther Sampson
ALL-EXON-ARRAYS
NovartisPharma AG
FFPE samples & MMI Laser Microdissection
Ludger Klein-Hitpass
Rosemarie Walter & James Eliason
Asterand plc
FFPE samples
Institute forCell Biology(Tumor Research)
Array Facility, Essen, Germany
Bodo Brunner
Core Facility / FACS -Sorted Stem Cells
Large and small samples
aventis-sanofi AG, Frankfurt, Germany
Craig P. Hunter & Ryan Baugh*
Renate Burgemeister & Ulrich Sauer
Dept. of Molecular andCellular Biology
Harvard University, Cambridge, USA
PALM, Bernried, Germany
Picogram RNA Samples
* now: [email protected]
Chris Shepherd & David Hudson
The Cancer Research Institute, U.K.
Degraded RNA Samples
Laser Microdissection / RNA isolation /
RNA amplification / Microarrays
Edda Stoinski, MMI Zürich, Switzerland
Wolfgang Kemmner, MDC Berlin, Germany
Laser Microdissection / RNA isolation /
RNA amplification / Microarrays
Otto Hagenbüchle
ISREC, Epalinges, Switzerland
Large and Small Samples
[email protected]
www.amp-tec.com